Elsevier

Biochimie

Volume 84, Issue 11, 1 November 2002, Pages 1047-1059
Biochimie

Complex restriction enzymes: NTP-driven molecular motors

https://doi.org/10.1016/S0300-9084(02)00020-2Get rights and content

Abstract

Survival is assuredly the prime directive for all living organisms either as individuals or as a species. One of the main challenges encountered by bacterial populations is the danger of bacteriophage attacks, since infection of a single bacterium may rapidly propagate, decimating the entire population. In order to protect themselves against this acute threat, bacteria have developed an array of defence mechanisms, which range from preventing the infection itself via interference with bacteriophage adsorption to the cell surface and prevention of phage DNA injection, to degradation of the injected phage DNA. This last defence mechanism is catalysed by the bacterial restriction–modification (R–M) systems, and in particular, by nucleoside 5′-triphosphate (NTP)-dependent restriction enzymes, e.g. type I and type III R–M systems or the modification-dependent endonucleases. Type I and type III restriction systems have dual properties. They may either act as methylases and protect the host’s own DNA against restriction by methylating specific residues, or they catalyse ATP-dependent endonuclease activity so that invading foreign DNA lacking the host-specific methylation is degraded. These defence mechanism systems are further complemented by the presence of methylation-dependent, GTP-dependent endonucleases, that restricts specifically methylated DNA. Although all three types of endonucleases are structurally very different, they share a common functional mechanism. They recognise and bind to specific DNA sequences but do not cleave DNA within those target sites. They belong to the general class of DNA motor proteins, which use the free energy associated with nucleoside 5′-triphosphate hydrolysis to translocate DNA so that the subsequent DNA cleavage event occurs at a distance from the endonuclease recognition site. Moreover, DNA cleavage appears to be a random process triggered upon stalling of the DNA translocation process and requiring dimerisation of the bound endonucleases for a concerted break of both DNA strands. In this review, we present a detailed description and analysis of the functional mechanism of the three known NTP-dependent restriction systems: type I and type III restriction–modification enzymes, as well as the methylation-dependent McrBC endonuclease.

Introduction

Natural selection is one of the primary forces in nature and bacterial survival depends heavily on the cells’ ability to protect themselves against bacteriophage attacks/infections. One of the mechanisms that bacteria have evolved for this purpose is nucleoside triphosphate-dependent (NTP) restriction enzymes (for previously published reviews, see [1], [2], [3], [4], [5]). Restriction–modification (R–M) enzymes have the dual opposing functions of: (i) protecting the host DNA against restriction by methylating the DNA within specific target sites, and (ii) “restricting”, i.e. degrading any unmodified DNA that may enter the cell. Based on their particular subunit structures and co-factor requirement, R–M enzymes have been classified into three main groups. Type II R–M enzymes comprise separate endonucleases and methylases that act independently from each other and have as a sole common factor the ability to target for cleavage or methylation, respectively, a single specific DNA sequence. This recognition site is typically a 4–8 bp palindromic sequence. Type II restriction enzymes have simple co-factor requirements: restriction depends on the presence of Mg2+, and modification requires S-adenosyl-methionine (AdoMet). Type I R–M systems are hetero-oligomeric enzymes constituted of three subunits: HsdR (host specificity for DNA, restriction), HsdM (modification or methylation) and HsdS (specificity) encoded by the hsd genes (hsdR, hsdM and hsdS, respectively) encoded contiguously on the bacterial genome. Type I restriction enzymes are present in two different oligomeric forms in vivo. The M2S form containing two HsdM and one HsdS subunits catalyses methylation of the DNA in the presence of the co-factors AdoMet and Mg2+ [6], [7], [8], [9], whereas the multimer R2M2S (M2S + two HsdR subunits) requires the presence of three co-factors, AdoMet, Mg2+ and ATP, for restriction of unmethylated DNA. Although it was originally thought that type I restriction enzymes might be restricted to the Enterobacteriaceae [1], [3], the genome sequencing projects developed in the last few years have revealed their presence in a large number of bacterial genera. A list of all type I restriction enzymes identified so far has been compiled and is maintained on the REBASE web site [10]. Type III R–M enzymes are composed of two subunits, products of the mod and res genes [11], [12]. The Mod subunit forms a stable dimer that acts as an independent modification methylase in the presence of AdoMet. In contrast, the Res subunit has no enzymatic activity when not complexed with Mod. The complex Res2Mod2 mediates DNA cleavage in the presence of ATP and the reaction is stimulated by AdoMet. This bacterial defence mechanism array is complemented by modification-dependent restriction (MDR) systems that specifically recognise and cut modified DNA. These MDR endonucleases have no associated methylase. The best-characterised member of MDR enzymes, McrBC, is a two-subunit complex that has a GTP-hydrolysis activity and a strict requirement for methylated DNA as a substrate. This review concentrates on the ATP-dependent type I and type III restriction–modification enzymes, on the GTP-hydrolysing McrBC complex and their respective functions.

Section snippets

Type I R–M enzymes

Type I R–M enzymes are multi-functional complexes. They catalyse many different functions such as recognition of and binding to a specific DNA sequence, methylation of specific adenine residues within that same sequence, DNA translocation coupled to ATP hydrolysis and, finally, DNA cleavage. Although the subunits of type I R–M enzymes cannot act independently from the complex, each of the functions enumerated above can be attributed specifically to one of the three subunits. The recognition

Type III R–M enzymes

Type III restriction–modification enzymes are hetero-oligomeric, multi-functional enzymes composed of the Mod subunit responsible for site-specific methylation of the DNA and the Res subunit that is able, after formation of a (Mod)2(Res)2 complex, to translocate and cleave DNA in an ATP-dependent fashion. The recognition sequence of type III enzymes is asymmetric, uninterrupted and 5–6 bp in length. Cleavage requires the presence of inversely oriented recognition sites and occurs at a distance

Modification-dependent restriction enzymes

Historically, modification-dependent restriction (MDR) endonucleases represent the first restriction systems ever described [86], predating even the well-known, classical phage assay experiments that led to the identification of Ecoli type I R–M systems [87], [88]. The first studied MDR enzymes were aimed at T-even phages. They exhibited restriction activity against non-glycosylated 5-hydroxymethylcytosine (HMC)-DNA in T-even phages, and were therefore named Rgl for restricts glucose-less

Conclusion

In this review, we have described three bacterial restriction systems that belong to the general class of DNA motor proteins, which use the free energy associated with nucleoside 5′-triphosphate to translocate DNA and thus trigger DNA cleavage randomly, away from the recognition site. Of the three different restriction systems described here, two were restriction–modification systems and utilised ATP, i.e. type I and type III enzymes, and one, the modification-dependent endonuclease, McrBC,

Acknowledgements

Work from this laboratory was supported by the Swiss National Science Foundation.

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